Chikungunya virus (CHIKV) recently emerged from Africa into an urban human-mosquito cycle to cause millions of cases of highly debilitating, often chronic arthralgic disease. Dozens of CHIKV importations via viremic travelers underscore the risk of CHIKV to the Western hemisphere. There is no effective treatment for chikungunya fever (CHIK), which can be fatal, so an effective vaccine is needed. For use in third world, endemic locations, an ideal vaccine must be inexpensive to manufacture and induce rapid, long-lived immunity after a single dose. To be used safely in nonendemic locations, the vaccine must also be incapable of infecting mosquito vectors in the event that viremia occurs in a vaccinee with impaired host defense. To achieve these goals, we developed novel, innovative approaches to stable alphavirus attenuation that retain immunogenicity yet eliminate the possibility of mosquito infection. Using a picornavirus internal ribosome entry site (IRES) that functions poorly in insect cells, combined with inactivation of the alphavirus subgenomic promoter that drives overexpression of the structural proteins, we produced a live CHIKV vaccine strain that combines the proven advantages of live-attenuated vaccines, yet which should be more stably attenuated than traditional approaches. This CHIKV vaccine is highly attenuated yet immunogenic in mouse models, and is incapable of replicating in mosquito cells. In this project, we will further develop this CHIK vaccine candidate in preparation for an investigational new drug application and Phase 1 human studies, using the following specific aims: 1. Determine the mechanism of vaccine attenuation by analyzing levels of RNA replication, protein expression and translational efficiency in vitro and patterns of replication and spread in vivo. 2. Define correlates of protective immunity and determine components of the innate and adaptive immune response required to control vaccine replication. 3. Optimize the vaccine candidate for production in Vero cells. 4. Develop and characterize GMP master virus seed and working virus seed stocks. 5. Develop quality control and release tests and manufacture a preclinical GMP lot of the vaccine. 6. Test the vaccine in nonclinical GLP studies for safety. 7. IND submission and manufacture of vaccine lots for Phase 1 clinical testing. The innovative product of this collaboration between academic and industrial partners will have dramatic impacts in Asia and Africa where CHIK causes both severe health effects and economic hardship, and will greatly reduce the risk of CHIKV importation and establishment in the Western Hemisphere. In addition, the improved mechanistic understanding of its attenuation and further development of our IRES-based vaccine platform technology will allow us to rapidly generate predictably safe and effective vaccines against other alphaviruses that represent biodefense threats and/or that have the potential that emerge in the future.